scholarly journals Bordetella parapertussis PagP Mediates the Addition of Two Palmitates to the Lipopolysaccharide Lipid A

2014 ◽  
Vol 197 (3) ◽  
pp. 572-580 ◽  
Author(s):  
L. E. Hittle ◽  
J. W. Jones ◽  
A. M. Hajjar ◽  
R. K. Ernst ◽  
A. Preston
Keyword(s):  
Lipid A ◽  
Mutational Analysis ◽  
Mass Spectrometry Analysis ◽  
Toll Like Receptor 4 ◽  
Membrane Function ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Proinflammatory Responses ◽  
Acyl Chains ◽  
Increased Sensitivity

Bordetella bronchisepticaPagP (PagPBB) is a lipid A palmitoyl transferase that is required for resistance to antibody-dependent complement-mediated killing in a murine model of infection.B. parapertussiscontains a putativepagPhomolog (encodingB. parapertussisPagP [PagPBPa]), but its role in the biosynthesis of lipid A, the membrane anchor of lipopolysaccharide (LPS), has not been investigated. Mass spectrometry analysis revealed that wild-typeB. parapertussislipid A consists of a heterogeneous mixture of lipid A structures, with penta- and hexa-acylated structures containing one and two palmitates, respectively. Through mutational analysis, we demonstrate that PagPBPais required for the modification of lipid A with palmitate. While PagPBBtransfers a single palmitate to the lipid A C-3′ position, PagPBPatransfers palmitates to the lipid A C-2 and C-3′ positions. The addition of two palmitate acyl chains is unique toB. parapertussis. Mutation ofpagPBParesulted in a mutant strain with increased sensitivity to antimicrobial peptide killing and decreased endotoxicity, as evidenced by reduced proinflammatory responses via Toll-like receptor 4 (TLR4) to the hypoacylated LPS. Therefore, PagP-mediated modification of lipid A regulates outer membrane function and may be a means to modify interactions between the bacterium and its human host during infection.

scholarly journals Nonribosomal Peptide Synthetase GenespesLandpes1Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

2012 ◽  
Vol 78 (9) ◽  
pp. 3166-3176 ◽  
Author(s):  
Karen A. O'Hanlon ◽  
Lorna Gallagher ◽  
Markus Schrettl ◽  
Christoph Jöchl ◽  
Kevin Kavanagh ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Mass Spectrometry Analysis ◽  
Infection Model ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Peptide Synthetases ◽  
Increased Sensitivity

ABSTRACTThe identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway inA. fumigatus. Deletion of eitherpesL(ΔpesL) orpes1(Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion ofpesLresulted in severely reduced virulence in an invertebrate infection model (P< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster forA. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in bothA. fumigatuswild-type and ΔpesLstrains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesisin vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.

scholarly journals Coidentification of mcr-4.3 and blaNDM-1 in a Clinical Enterobacter cloacae Isolate from China

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Bhakti Chavda ◽  
Jingnan Lv ◽  
Mengyun Hou ◽  
Kalyan D. Chavda ◽  
Barry N. Kreiswirth ◽  
...  
Keyword(s):  
Lipid A ◽  
Susceptibility Testing ◽  
Enterobacter Cloacae ◽  
Mass Spectrometry Analysis ◽  
Ionization Mass ◽  
The Novel ◽  
Laser Desorption Ionization ◽  
Content Type ◽  
Spectrometry Analysis ◽  
High Level

ABSTRACT We describe the first report of a clinical colistin-resistant ST84 Enterobacter cloacae isolate coharboring mcr-4.3 (previously named mcr-4.2) and blaNDM-1 from a patient in China. The blaNDM-1-harboring IncX3 plasmid and the novel mcr-4.3-harboring ColE plasmid were completely sequenced. Although this isolate showed a high level of resistance to colistin, mcr-4.3 plasmid transformation, gene subcloning, susceptibility testing, and lipid A matrix-assisted laser desorption ionization mass spectrometry analysis indicated that mcr-4.3 itself does not confer resistance to colistin.

Mass spectrometry analysis of intact Francisella bacteria identifies lipid A structure remodeling in response to acidic pH stress

Biochimie ◽  
2017 ◽  
Vol 141 ◽  
pp. 16-20 ◽  
Author(s):  
Camille B. Robert ◽  
Michael Thomson ◽  
Alain Vercellone ◽  
Francesca Gardner ◽  
Robert K. Ernst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid A ◽  
Mass Spectrometry Analysis ◽  
Acidic Ph ◽  
Spectrometry Analysis ◽  
Ph Stress ◽  
Lipid A Structure

scholarly journals Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

2010 ◽  
Vol 192 (8) ◽  
pp. 2044-2052 ◽  
Author(s):  
Jyl S. Matson ◽  
Hyun Ju Yoo ◽  
Kristina Hakansson ◽  
Victor J. DiRita
Keyword(s):  
Antimicrobial Peptides ◽  
Vibrio Cholerae ◽  
Lipid A ◽  
Polymyxin B ◽  
Mass Spectrometry Analysis ◽  
Peptide Antibiotic ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Antimicrobial Peptide Resistance ◽  
El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

scholarly journals Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

2012 ◽  
Vol 80 (12) ◽  
pp. 4333-4343 ◽  
Author(s):  
Barak Hajaj ◽  
Hasan Yesilkaya ◽  
Rachel Benisty ◽  
Maayan David ◽  
Peter W. Andrew ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mass Spectrometry Analysis ◽  
Activity Levels ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Thiol Peroxidase ◽  
Fine Tune

ABSTRACTStreptococcus pneumoniaeis an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth,S. pneumoniaeproduces high levels of H2O2. SinceS. pneumoniaelacks catalase, the question of how it controls H2O2levels is of critical importance. Thepsalocus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase,tpxD. This study shows thattpxDencodes a functional thiol peroxidase involved in the adjustment of H2O2homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2efficiently. However,in vivoexperiments revealed that TpxD detoxifies only a fraction of the H2O2generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58undergoes selective oxidationin vivo, under conditions where H2O2is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesisin vitrowere significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2resulted intpxDupregulation, whilepsaBCAexpression was oppositely affected. However, the challenge of ΔtpxDmutants with H2O2did not affectpsaBCA, implying that TpxD is involved in the regulation of thepsaoperon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.

scholarly journals Dissection of the Role of Pili and Type 2 and 3 Secretion Systems in Adherence and Biofilm Formation of an Atypical Enteropathogenic Escherichia coli Strain

2013 ◽  
Vol 81 (10) ◽  
pp. 3793-3802 ◽  
Author(s):  
Rodrigo T. Hernandes ◽  
Miguel A. De la Cruz ◽  
Denise Yamamoto ◽  
Jorge A. Girón ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Coli Strain ◽  
Mass Spectrometry Analysis ◽  
Secretion Systems ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Rigid Rod

ABSTRACTAtypical enteropathogenicEscherichia coli(aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2eaemutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion offimAin 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2sslEmutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a doubleeae espAmutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2eaemutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

scholarly journals Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Hongming Zhang ◽  
Bettina A. Buttaro ◽  
Derrick E. Fouts ◽  
Salar Sanjari ◽  
Bradley S. Evans ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Amino Acid Identity ◽  
Lytic Infection ◽  
Mass Spectrometry Analysis ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

scholarly journals Modification of Lipooligosaccharide with Phosphoethanolamine by LptA in Neisseria meningitidis Enhances Meningococcal Adhesion to Human Endothelial and Epithelial Cells

2008 ◽  
Vol 76 (12) ◽  
pp. 5777-5789 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Russel W. Carlson ◽  
Artur Muszynski ◽  
Biswa Choudhury ◽  
Kwang Sik Kim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neisseria Meningitidis ◽  
Lipid A ◽  
Inner Core ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Epithelial Cell Lines

ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.

scholarly journals Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

2015 ◽  
Vol 197 (9) ◽  
pp. 1668-1680 ◽  
Author(s):  
Sarah Siu ◽  
Anna Robotham ◽  
Susan M. Logan ◽  
John F. Kelly ◽  
Kaoru Uchida ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mass Spectroscopy ◽  
Specific Antigen ◽  
Mass Spectrometry Analysis ◽  
Methanococcus Maripaludis ◽  
Western Blots ◽  
Bacterial Gene ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Type Iv

ABSTRACTMethanococcus maripaludishas two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354andmmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions inmmp0350,mmp0351,mmp0352, ormmp0353were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352strain confirmed a glycan with only GalNAc, suggestingmmp0350tommp0353were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr).M. maripaludismmp0350,mmp0351,mmp0352,mmp0353, andmmp0357are proposed to be functionally equivalent toPseudomonas aeruginosawbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of theP. aeruginosapathway withmmp0357supports this hypothesis.IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins ofMethanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway.

scholarly journals A Unique Glycine-Rich Motif at the N-terminal Region of Bamboo mosaic virus Coat Protein Is Required for Symptom Expression

2010 ◽  
Vol 23 (7) ◽  
pp. 903-914 ◽  
Author(s):  
Ping Lan ◽  
Wen-Bin Yeh ◽  
Chih-Wei Tsai ◽  
Na-Sheng Lin
Keyword(s):  
Mass Spectrometry ◽  
Mosaic Virus ◽  
Mutational Analysis ◽  
Plant Viruses ◽  
Site Directed Mutagenesis ◽  
Mass Spectrometry Analysis ◽  
Coat Proteins ◽  
Long Distance ◽  
Spectrometry Analysis ◽  
Local Lesions

The coat proteins (CP) of many plant viruses are multifunctional proteins. We used N-terminal sequencing and mass spectrometry/mass spectrometry analysis to identify a truncated form of the Bamboo mosaic virus (BaMV) CP missing the N-terminal 35 amino acids (N35). The N35 region is unique in the potexviruses by its containing a glycine-rich motif (GRM) not present in databases but highly conserved among BaMV isolates. Results from site-directed mutagenesis and deletion mutational analysis showed that loss of this region converted necrotic local lesions to chlorotic local lesions on Chenopodium quinoa leaves. Furthermore, this region is required for successful development of mosaic symptoms on Nicotiana benthamiana leaves but is dispensable for BaMV replication and cell-to-cell and long-distance movement as well as virion assembly. This unique GRM-containing region of BaMV CP may be a symptom determinant in specific hosts.

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